北美海蓬子盐胁迫条件下抑制差减文库构建与分析

Construction and Analysis of Differentially Expressed cDNA Library of Salicornia Bigelovii by Using Suppression Subtractive Hybridization

北美海蓬子(Salicornia Bigelovii Torr.)是一种耐盐性极强的真盐生植物,在200mmol/L的Na+浓度下生长,其高度和含汁量是5mmol/LNa+浓度下生长的2倍和2.5倍。为了研究北美海蓬子耐盐的机理和分离耐盐新基因,我们构建了在盐胁迫条件下北美海蓬子基因表达差异的SSH文库。以北美海蓬子种子播种于0mmol/LNaCl MS液体培养基上幼苗(0号材料)的总RNA为Driver,200mmol/LNaCl诱导24h幼苗(2号材料)的总RNA为Tester,运用SMART PCR cDNA合成技术分别合成全长cDNA。将两种cDNA群体进行抑制差减杂交,用T/A法分别构建了盐胁迫相关基因的正向差减文库(205个克隆子)和反向差减文库(235个克隆子)。通过PCR和反式NorthernDot-Blot消除假阳性和重复克隆子,得到在盐胁迫下特异表达的阳性克隆85个。对测序结果正常的81个克隆进行BLASTn分析,其中有70个cDNA序列与已经报道的其它物种的核酸序列具有较高的同源性;另外11个cDNA序列未找到有较高相似性的已知序列。81个cDNA序列中没有与北美海蓬子中已报道的核酸序列有较高的相似性,因此,所有81个克隆序列都是北美海蓬子中的新基因。

Salicornia Bigelovii Torr. is a high salt-tolerant euhalophyte. Plants growing in 200 mmol/L NaCl are 2-flod tall and more than 2.5-flod succulent of plants growing in 5 mmol/L NaCl. In order to study the mechanism of salt tolerance and isolate new genes related to salt tolerance in S. Bigelovii Torr., the SSH library has been constructed. The completed cDNAs were synthesized by using the SMART PCR cDNA technique, in which mRNAs isolated from the plants growing on MS liquid medium with 0 mmol/L NaCl （sample 0） as driver, and the mRNA isolated from the plants induced with 200 mmol/L NaCl for 24 h （sample 2） as tester. The forward SSH library containing 205 clones and the backward SSH library containing 235 were constructed by using the T/A clone method. Finally, 85 positive clones with different segments have been selected by using PCR and reverse northern blot. 81 clones of the above 85 clones, which were sequenced normally, have been analyzed by BLASTn. The results showed that 70 clones are homology to the reported genes from another plants; 11 clones are very low or no homology to any reported genes. All of the 81 clones have no homology to the genes reported in S. Bigelovii Torr., thus they are likely to be new genes.